Biomedical subjects
Publications and source records attributed to W L Nicholson.
Borrelia anserina (Sakharoff) was successfully grown in a liquid medium (Barbour-Stoenner-Kelly) for 39 passages. By the 12th serial passage in medium, infectivity of B anserina for chicks was lost. Electron microscopy did not reveal structural differences between non-infective and infective cultured organisms. Changes in the protein profiles were found by electrophoresis as the organisms were passed in culture.
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The gene encoding glucose dehydrogenase (gdh) is part of an operon whose expression is transcriptionally activated specifically in the developing forespore of Bacillus subtilis at stage III of sporulation. The in vivo startpoint of gdh transcription was determined using primer extension analysis. Deletion mapping and site-specific mutagenesis experiments indicated that the region responsible for regulated expression of gdh in vivo was limited to the "-35" and "-10" regions preceding the transcriptional start site. RNA polymerase containing omega G (E omega G) transcribed gdh in vitro with a start site identical to that found in vivo, and transcription of gdh by E omega G in vitro also did not require any specific sequences upstream from "-35" region. These results suggest that the appearance of E omega G in the forespore at stage III of sporulation is sufficient to cause temporal and compartment-specific expression of the gdh operon.
Antigenic cross-reactivity between Borrelia burgdorferi and Borrelia anserina was studied using mouse immune sera and monoclonal antibodies. With immune sera, significant cross-reactivity between B. burgdorferi and B. anserina was demonstrated by indirect immunofluorescent assay. In immunoblots, most of the cross-reactivity was shown to be associated with the periplasmic flagella. Using monoclonal antibodies in immunoblots, it was shown that B. burgdorferi and B. anserina shared at least two flagellar epitopes, one of which was not shared with Borrelia hermsii or Borrelia coriaceae. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of whole cell lysates and the use of a species-specific monoclonal antibody (H5332) which reacts with a major outer surface protein (Osp A) of B. burgdorferi readily differentiated the two species at the molecular level.
During sporulation in Bacillus subtilis, expression of the genes sspA, sspB, sspC, sspD, and sspE, which encode a family of small, acid-soluble spore proteins, as well as of the spoVA and gdh operons is transcriptionally activated at stage III of sporulation only in the forespore compartment. Transcription of these genes is mediated by RNA polymerase containing sigma G (E sigma G), the product of the sigG gene, which is itself expressed at stage III in the developing forespore. We have determined the 5' ends of transcripts generated both in vivo and in vitro by the action of E sigma G on various genes of B. subtilis and other bacilli. The 5' ends of the in vivo and in vitro mRNAs were found to coincide and were therefore considered to define the transcription initiation sites for the genes examined. We identified highly homologous DNA sequences centered at 35 and 10 base pairs preceding the transcriptional start sites of the genes examined. Consequently, we propose that these sequences define a class of promoters recognized only by E sigma G which allow transcription of genes expressed uniquely at stage III in the developing forespore.
Immunoblots were used to study the immunoglobulin G response to Borrelia burgdorferi in experimentally and naturally exposed dogs. Adsorption studies confirmed that the antibodies were specific for B. burgdorferi. Experimentally exposed dogs were asymptomatic. Naturally exposed dogs included both asymptomatic animals and animals showing signs compatible with Lyme disease. Naturally exposed dogs were from four geographic regions of the country. No differences were detected between immunoblot patterns of naturally exposed symptomatic or asymptomatic dogs from different areas of the country. The immunoblot patterns obtained with sera from experimentally exposed dogs were different from those obtained with sera from naturally exposed dogs and were characterized by reactivity to fewer and different protein bands. Immunoblot analysis using an OspA-protein-producing Escherichia coli recombinant showed that experimentally exposed dogs produced antibodies to OspA, whereas naturally exposed dogs did not. Modifications of the immune response over time, different routes of antigen presentation, and strain variation are factors postulated to account for the observed differences.
Adult Beagles were used to evaluate clinical signs and serologic response after inoculation with, or exposure to, Borrelia burgdorferi. An indirect immunofluorescent assay (IFA) and 2 ELISA were used to monitor the serologic response to B burgdorferi. Feeding infected ticks on 4 dogs (group 1) failed to cause seroconversion, and SC inoculation with 500 organisms caused minimal seroconversion in 2 of 4 dogs (group 2). At 56 days, approximately 3.01 X 10(8) B burgdorferi organisms were injected IV into group-1 dogs, and intraperitoneally into group-2 dogs. A control group of 4 dogs (group 3) had noninfected ticks feed on them, and then were given IV injection of physiologic saline solution. Increases in immunoglobulin M (IgM) titers were detected in 2 of 4 group-2 dogs approximately 7 days after the initial exposure. These titers returned to negligible values 20 days later. Immunoglobulin G titers increased approximately 10 days after the initial exposure and were mildly increased 56 days later, when dogs were exposed a second time. Both the IV and intraperitoneal injections (second exposures) resulted in increased IgM titers, which in both groups eventually returned to preexposure values after approximately 2 months. Immunoglobulin G titers increased within a week after the second exposure, and in 3 dogs monitored for 8 months, returned to negligible values after the 8-month period. One control dog had a slightly increased IgG titer 24 days after the second inoculation. The possibility of urine transmission is suggested. Clinical status, hemograms, serum biochemical profiles, ECG and results of urinalyses remained normal throughout the study.(ABSTRACT TRUNCATED AT 250 WORDS)
The amyR1 locus controls the regulated transcription of amyE, the structural gene encoding alpha-amylase in Bacillus subtilis. Transcription of amyE is activated in early stationary phase cells, and can be repressed by rapidly metabolized carbon sources such as glucose. Transcription of amyE initiates in vitro from a promoter recognized by the major vegetative form of RNA polymerase, E sigma 43. S1 nuclease mapping of in-vivo amylase transcripts suggests that this promoter is also used in vivo. Two independently isolated cis-acting mutations, gra-5 and gra-10, which abolish glucose-mediated repression of amylase synthesis without altering temporal activation, were determined by DNA sequencing to result from a G.C to A.T transition at a position located five base-pairs downstream from the start site of transcription. While this is the first example of a site involved in catabolite repression of gene expression in a Gram-positive micro-organism, the region surrounding the gra mutations shows considerable homology to certain cis-acting regulatory loci in Escherichia coli, suggesting that such sequences have been evolutionarily conserved.
Decoyinine, an inhibitor of GMP synthetase, allows sporulation in Bacillus subtilis to initiate and proceed under otherwise catabolite-repressing conditions. The effect of decoyinine on alpha-amylase synthesis in B. subtilis, an event which exhibits regulatory features resembling sporulation initiation, was examined. Decoyinine did not overcome catabolite repression of alpha-amylase synthesis in a wild-type strain of B. subtilis but did cause premature and enhanced synthesis in a mutant strain specifically blocked in catabolite repression of alpha-amylase synthesis. Decoyinine had no effect on alpha-amylase enzymatic activity. Thus, it appears that the catabolite control mechanisms governing alpha-amylase synthesis and sporulation in B. subtilis differ in their responses to decoyinine and hence must consist at least partially of separate components.
Three cis-acting alleles (gra-10, gra-5, and amyR2) of the Bacillus subtilis amyR promoter locus each cause catabolite repression-resistance of amyE-encoded alpha-amylase synthesis. The gra-10, gra-5, and amyR2 alleles were transferred from the chromosomes of their respective hosts to a plasmid carrying the amyR1-amyE+ gene by the process of gene conversion which is carried out during transformation of competent B. subtilis by plasmid clones carrying homologous DNA. The cloned amyR promoter regions containing the gra-10 and gra-5 mutations were shown to confer catabolite repression-resistance in cis to the synthesis of chloramphenicol acetyltransferase encoded by the cat-86 indicator gene when subcloned into the promoter-probe plasmid pPL603B. Implications concerning both the regulation of amyR utilization and the process of gene conversion in B. subtilis are discussed.
The amyR1 region controls the regulated expression of the Bacillus subtilis 168 amylase gene amyE. When cloned into the B. subtilis promoter-cloning plasmid pPL603, amyR1 has been shown to activate expression of the promoter-indicator gene cat-86. In this chimeric plasmid, p5' alpha B10, cat-86 expression was maximal in stationary phase B. subtilis cells and cat-86 expression was repressible by glucose. Both these properties are similar to the regulated expression of the B. subtilis amyE gene. In addition, cat-86 expression in p5' alpha B10 was inducible with chloramphenicol (Cm). The inducibility phenotype of cat-86 has been shown to be independent of the promoter that is used to activate the gene, and inducibility has been suggested to result from the presence of a pair of inverted-repeat sequences that span the ribosome-binding site (RBS) for cat-86. A spontaneous deletion mutant of p5' alpha B10 was isolated, p5' alpha B10 delta 1, in which cat-86 expression was constitutive with respect to Cm, but the basic pattern of amyR1-directed regulation of cat-86 was intact. The rightward deletion endpoint was within the upstream member of the pair of inverted repeats that immediately precede cat-86. This result is therefore consistent with the role proposed for the inverted repeats in Cm inducibility. The leftward endpoint of the deletion is within the amyR1 region and thus allows a more precise determination of the functional domain of amyR1.
Bacillus subtilis 168GR10 was shown to contain a mutation, gra-10, which allowed normal temporal activation of alpha-amylase synthesis in the presence of a concentration of glucose that is inhibitory to activation of amylase synthesis in the parent strain, 168. The gra-10 mutation was mapped by phage PBS-1-mediated transduction and by transformation to a site between lin-2 and aroI906, very tightly linked to amyE, the alpha-amylase structural gene. The gra-10 mutation did not pleiotropically affect catabolite repression of sporulation or of the synthesis of extracellular proteases or RNase and was unable to confer glucose-resistance to the synthesis of chloramphenicol acetyltransferase encoded by the cat-86 gene driven by the amyE promoter region (amyR1) inserted into the promoter-probe plasmid pPL603B. It therefore appears that gra-10 defines a cis-regulatory site for catabolite repression, but not for temporal activation, of amyE expression. The evidence shows that temporal activation and glucose-mediated repression of alpha-amylase synthesis in B. subtilis 168 are distinct phenomena that can be separated by mutation.
The purpose of the paper is twofold: first, to describe the aspects of quantitative microscopy where statistical ideas are being applied today; and second, to describe some ways in which more complete use of statistics could improve quantitative microscopy. The typical estimation problem in quantitative microscopy is described with emphasis on the modelling of variability in estimation. A Poisson field model for imbedded particulates is used to illustrate the value of theoretical treatments of variability. Sampling, estimation and multivariate analysis are cited as areas of statistics presently used by quantitative microscopists. The potential role of statistics in quantitative microscopy is discussed. Examples from particle sizing and characterization of alveolar lung structure illustrate the value of statistical ideas. Automatic image analysers, while making the collection of large amounts of data feasible, have complicated statistical estimation. An investigation of the size distribution of random chord lengths through alveolar chambers illustrates how statistical methodology must be modified for valid inference.
Meningitis is a rare complication following stapedectomy. Most cases are preceeded by a fistula in the oval window. Although fatalities have been reported, most cases respond to parenteral and intrathecal antibiotics if diagnosed and treated early. Gram-positive organisms are the usual pathogens, but Gram-negative organisms may be found, especially as a superinfection following antibiotics administered prophylacticly or for the treatment of otitis media or mastoiditis. A case of Pseudomonas aeruginosa meningitis five weeks after stapedectomy, successfully treated with gentamicin, is reported. The discussion included the diagnosis of post-stapedectomy fistula and meningitis and current trends in treatment.
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